Figure 9 shows the contribution of the membrane and late endosome components, on ERK activation at EGF concentrations ranging from 25 ng/ml to 100 ng/ml. Interestingly, the signaling via the late endosomal component is insensitive to the variation of EGF concentrations under this condition. In contrast, signaling through the two membrane components is substantially altered by varying EGF doses, specifically when EGF is reduced from 40 ng/ml to 25 ng/ml. Supplementary Figure S5 further shows the relative sensitivity of these two sub-pathways at high EGF dose �C the sensitivity of membrane subpathway is almost 4 times the sensitivity of endosomal subpathway. These results are consistent with the prediction using principle component analysis that receptor internalization and endosomal signaling are important features regulating signal output at lower EGF doses. Through ligand-induced receptor activation, any changes in the EGF concentrations could lead to altered levels of activated EGFR on the cell surface, thus affecting its downstream signaling via both the membrane components and the late endosomal component. We therefore hypothesize that the apparent difference in their ligand-sensitivity could be influenced not just by the scaffolds alone but most likely via their relative concentrations and interplay with other immediate regulators such as the Cbl-CIN85 and Endophilin A1. The KSR-scaffolded pathway and the conventional pathway are sensitive to EGF stimulation and their combined Everolimus effects on ERK activation are synergistic. When the KSR level is high, the sensitivity of this combined pathway remains low in the presence of low concentration of Cbl-CIN85 while such sensitivity can be increased with increasing levels of Endophilin A1 if the amount of Cbl-CIN85 becomes high. However, reduced KSR level already presents high sensitivity that is independent of the levels of Endophilin A1. In contrast, the ERK activation by MP1-scaffolded pathway is additive to that of KSR but it shows little ligand-sensitivity under high levels of EGF stimulation. Such inert sensitivity can, however, be reversed in part by increasing level of Endophilin A1 while keeping the level of Cbl-CIN85 low or by increasing level of Cbl-CIN85 while keeping the level of Endophilin A1 low. Thus, this current study extends the observations of others, thereby suggesting that the process of endocytosis plays a prominent role in regulating signal output sensitivity in response to different EGF dosages. Since Cbl-CIN85 and Endophilin A1 promote endocytosis of activated EGF receptors and Vorinostat HDAC inhibitor facilitate trafficking of the signaling complex to late endosomes, we went on to analyze the concomitant modulation of receptor endocytosis in addition to the dynamics of ERK activity. Our analyses showed that, when the levels of scaffold proteins KSR and/or MP1 was either high, low or optimal, the sensitivity of endocytosed EGFR increased with increasing concentrations of Endophilin A1, if only when Cbl-CIN85 was present at high levels.